Immunology and Cell Biology (2007) 85, 532–537 & 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00 www.nature.com/icb
ORIGINAL ARTICLE
Interleukin-1a regulates antimicrobial peptide expression in human keratinocytes Mika Bando1, Yuka Hiroshima1, Masatoshi Kataoka2, Yasuo Shinohara3, Mark C Herzberg4, Karen F Ross4, Toshihiko Nagata1 and Jun-ichi Kido1 Human epidermis and epithelium serve as physiologic barriers to protect against noxious and infectious agents. Contributing to the defense against infection, epithelial cells express antimicrobial peptides (AMPs). The expression of AMPs in keratinocytes is generally regulated directly by bacteria and indirectly by proinflammatory cytokines. Bacteria may also regulate AMP expression by inducing keratinocyte expression of the autonomous proinflammatory cytokine, interleukin-1a (IL-1a). To test the hypothesis that AMP expression may be regulated by cell autonomous cytokines, we investigated the effect of IL-1a on the expression of AMPs in human keratinocytes (HaCaT cells) by microarray, northern blot, reverse transcriptase (RT)–PCR and western blot analyses. IL-1a increased expression of mRNA in a dose- and time-dependent manner specific for lipocalin 2, S100A8, S100A9 and secretory leukocyte protease inhibitor (SLPI) more than twofold relative to nonstimulated cells (control), and slightly upregulated S100A7 and b-defensin-2. Furthermore, the expression of lipocalin 2, S100A7, S100A8, S100A9 and SLPI proteins were upregulated by IL-1a. On the other hand, HaCaT cells expressed mRNA specific for other AMPs, including cystatin 3, adrenomedullin, RNase-7 and mucin 5, which were unaffected by IL-1a treatment. These results suggest that the autonomous keratinocyte cytokine, IL-1a, selectively upregulates the expression of AMPs which may modulate innate epithelial cell immunity in skin and mucosa. Immunology and Cell Biology (2007) 85, 532–537; doi:10.1038/sj.icb.7100078; published online 5 June 2007 Keywords: IL-1a; antimicrobial peptides; keratinocyte
The epidermis and epithelium contribute to host defense as a physical barrier against the invasion of many microbes. Defense is also augmented by epithelial innate immunity whereby keratinocytes produce antimicrobial peptides (AMPs), peptides with broad-spectrum antimicrobial activity, including b-defensins, calprotectin, secretory leukocyte protease inhibitor (SLPI) and lysozyme.1–3 The AMPs inhibit the adhesion and growth of microbes, but the spectrum of expressed AMPs varies in keratinocytes from different tissues and in health and disease. For example, human b-defensin-1 (hBD-1) is constitutively expressed in healthy skin and squamous mucosa, and also in inflamed epithelial tissues, but human b-defensin-2 and -3 (hBD-2 and -3) are abundantly expressed in inflamed epithelia.4–7 Calprotectin is a calcium-binding protein complex expressed in the suprabasal spinous cells of healthy mucosa but not in healthy skin.8–10 Calprotectin is generally upregulated in inflammation.11–14 SPLI is detected in healthy skin, renal tubules and salivary glands.15 While some AMPs are constitutively expressed in keratinocytes, other peptides are upregulated by inflammatory factors such as interleukin-1b
(IL-1b), tumor necrosis factor-a (TNF-a) and bacterial lipopolysaccharide (LPS).1,2,6 Interleukin-1a (IL-1a) is produced by keratinocytes, regulates keratinocyte differentiation and is expressed at higher levels in epidermis than other tissues.16 IL-1a and calcium induce keratinocyte differentiation and increase the expression of hBD-2 in several epithelial cell lines.17–19 IL-1a may also control innate cellular immunity in epithelial cells. Recently, IL-1a has been implicated in the control of innate immunity of epithelial cells by collaborating with interferon-g to coregulate antiviral activity.20 IL-1a and calcium also increase production of calprotectin, the antimicrobial S100 calcium-binding protein complex of S100A8 (MRP8; calgranulin A) and S100A9 (MRP14; calgranulin B) by human gingival keratinocytes as we have reported.3 These studies suggest that cytokines that regulate keratinocyte differentiation can also more generally control expression of AMPs. To test the hypothesis that AMP expression may be regulated by autonomous cytokines, we investigated the effect of IL-1a on the expression of AMP-specific mRNAs and proteins in human keratino-
1Department of Periodontology and Endodontology, Oral and Maxillofacial Dentistry, Division of Medico-Dental Dynamics and Reconstruction, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan; 2Health Technology Research Center, Advanced Industrial Science and Technology, Takamatsu, Kagawa, Japan; 3Division of Gene Expression, Institute for Genome Research, The University of Tokushima Graduate School, Tokushima, Japan and 4Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, MN, USA Correspondence: Dr Jun-ichi Kido, Department of Periodontology and Endodontology, Oral and Maxillofacial Dentistry, Division of Medico-Dental Dynamics and Reconstruction, Institute of Health, Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima, 770-8504, Japan. E-mail:
[email protected] Received 30 January 2007; revised 28 April 2007; accepted 4 May 2007; published online 5 June 2007
IL-1a regulates AMP expression M Bando et al 533
cytes (HaCaT cells) by microarray, northern blot, reverse transcriptase (RT)–PCR and western blot analyses.
Table 1 Genes differentially expressed in HaCaT cells in response to IL-1a
RESULTS Genes differentially expressed by IL-1a IL-1a increased HaCaT cell expression of 37 genes more than twofold compared to unstimulated controls and decreased expression of two genes to less than 0.5-fold of controls (Table 1). The most highly upregulated genes were protease inhibitor 3 (6.8-fold increase compared to control), lipocalin 2 (LCN2; 6.4-fold), chemokine (C-X-C motif) ligand 5 (5.4-fold), the epithelial-specific genes DD96 (5.4-fold), serum amyloid A1 (4.6-fold), S100A9 (4.2-fold) and S100A8 (4.0-fold). The expression of several other protease inhibitor genes was also increased, including serine proteinase inhibitors (3.7- and 2.9-fold) and SLPI (2.3-fold). In response to IL-1a, certain inflammatory factors showed 2.4- to 5.4-fold increased expression relative to control, including IL-1a and -1b, IL-8, chemokine (C-X-C motif) ligands and amyloid A. Several genes associated with cell growth or differentiation increased in expression in response to IL-1a by 2.1- to 2.5-fold, including T-cell leukemia/lymphoma 6 transcript variant TCL6a1, Ras association domain family 3 and leukemia inhibitory factor. Furthermore, IL-1a increased the expression of low-affinity Fc g-receptor IIb (CD32), an immunity-associated protein, about twofold. IL-1a also increased expression of genes for proline-rich protein and small proline-rich protein 1B, while inhibiting expression of genes for proline dehydrogenase (oxidase) 1, which degrades proline-rich proteins.
Gene
AMP genes regulated by IL-1a To learn if IL-1a regulates expression of AMP genes, 18 AMP genes with signal intensity greater than 100 were selected for comparison (Table 2). Among these AMP genes, the expression of LCN2, a major component in tears, and S100A8 and S100A9, were upregulated by IL-1a compared to control, and expression of SLPI, a cationic peptide, was increased by 2.3-fold. The expression of the S100 calcium-binding protein 7 (S100A7) and defensin-b 4 (DEFB4, hBD-2) increased about 1.5-fold in comparison to control. HaCaT cells also expressed 12 other putative AMP genes in a non-IL-1a-dependent manner, including cystatin C (CST3), adrenomedullin (ADM), RNase 7 (RNASE7), mucin 5 (MUC5B), b-defensins (DEFB1, DEFB103A and DEFB123), hepcidin antimicrobial peptide (HAMP), azurocidin 1 (AZU1), lysozyme 2 (LYZL2), seminal plasmin (PYY2) and dermcidin (DCD). Yet, other putative AMPs showed signal intensities in HaCaT cells less than 100, including cathelicidin, lactotransferrin and liver-expressed antimicrobial peptide 2 (data not shown). Validation of AMP gene expression upregulated by IL-1a Using RT-PCR and northern blot, expression of LCN2, S100A7, S100A8, S100A9 and SLPI genes were confirmed to increase in response to IL-1a stimulation of HaCaT cells (Figure 1). Likewise, the expression of DEFB4 gene, with less than twofold change, was confirmed to be upregulated by IL-1a using RT-PCR. However, IL-1a showed no apparent affect on the regulation of the expression of the AMP genes, CST3, ADM, RNASE7, MUC5B, DEFB103A, HAMP, DEFB1 and AZU1; the absence of affect was confirmed by RT-PCR (Figure 1a). The validation of AMP gene expression by northern blot or RT-PCR analysis was performed in at least three individual experiments and similar results were observed. Increased expression of AMPs, including S100A8, S100A9, LCN2, SLPI and S100A7, was seen as soon as 6 h of incubation with IL-1a and persisted for 72 h (Figure 1b). At 24 h, HaCaT cells were incubated with up to 100 mg/ml
Accession no.
Fold
Protease inhibitor 3, skin-derived Lipocalin 2
NM_002638 NM_005564
6.75 6.35
Chemokine (C-X-C motif) ligand 5 DD96
NM_002994 U21049
5.42 5.39
Serum amyloid A1, transcript variant 1 S100 calcium-binding protein A9
NM_000331 NM_002965
4.62 4.24
(S100A9) S100 calcium-binding protein A8
NM_002964
3.96
(S100A8) Serine proteinase inhibitor, clade B,
Upregulated genes
NM_006919
3.73
member 3 Proline-rich protein
M21539
3.63
Interleukin-8 Amyloid A-3 protein
NM_000584 ENST00000278207
3.21 3.14
Serum amyloid A2 mRNA full-length insert cDNA clone
NM_030754 AJ420586
3.03 3.01
Serine proteinase inhibitor, clade B, member 4
NM_002974
2.94
Small proline-rich protein 1B cDNA FLJ22893 fis, clone KAT04792
NM_003125 AK026546
2.90 2.72
Chemokine (C-X-C motif) ligand 1 Interleukin-1b
NM_001511 NM_000576
2.62 2.62
Calcium-binding protein 39-like cDNA FLJ38142 fis, clone
NM_030925 AK095461
2.56 2.51
D9OST2003108 Interleukin-1a
EUROIMAGE1998777
NM_000575
2.50
T-cell leukemia/lymphoma 6, transcript variant TCL6a1
NM_012468
2.45
Chemokine (C-X-C motif) ligand 6 Hypothetical protein FLJ13840
NM_002993 NM_024746
2.41 2.41
Chemokine (C-X-C motif) ligand 2 Secretory leukocyte protease inhibitor
NM_002089 NM_003064
2.38 2.32
Ras association (RalGDS/AF-6) domain family 3
NM_178169
2.26
Cytochrome P450, family 1, subfamily A, polypeptide 1
NM_000499
2.26
Hypothetical protein FLJ23231 G protein-coupled receptor 45
NM_025079 NM_007227
2.24 2.20
Superoxide dismutase 2, mitochondrial Normal mucosa of esophagus specific 1,
NM_000636 NM_032413
2.19 2.18
transcript variant 2 Leukemia inhibitory factor
NM_002309
2.13
Fc fragment of IgG, low-affinity IIb, receptor for CD32
NM_004001
2.13
Neuromedin B, transcript variant 1 Argininosuccinate synthetase
NM_021077 S73202
2.09 2.04
Testis-specific protein NYD-TSP1
NM_032567
2.04
AK074078
0.48
NM_016335
0.44
Downregulated genes mRNA for FKJ00149 protein Proline dehydrogenase (oxidase) 1
RNA was isolated from HaCaT cells after incubation with or without IL-1a (10 ng/ml) for 24 h. RNA was harvested as described in the Methods and mRNA expression was analyzed using Whole Human Genome Oligo Microarray (Agilent Technologies). The fold difference in mRNA expression level in IL-1a-stimulated HaCaT cells normalized to nonstimulated cells is shown.
Immunology and Cell Biology
IL-1a regulates AMP expression M Bando et al 534
Table 2 AMP gene expression by HaCaT cells in the presence of IL-1a Gene Lipocalin 2 (LCN2) S100 calcium-binding protein A9 (S100A9)
Accession no. NM_005564 NM_002965
RT-PCR
Cont. IL-1 α Cont. IL-1 α
Fold 6.35 4.24
RT-PCR
blotting Cont. IL-1α
Cont. IL-1α
LCN2
DEFB4
S100A9
CST3
HAMP
S100A8
ADM
DEFB1
SLPI
RNASE 7
AZU1
S100A7
MUC5B
GAPDH
S100 calcium-binding protein A8 (S100A8) Secretory leukocyte protease inhibitor (SLPI)
NM_002964 NM_003064
3.96 2.32
S100 calcium-binding protein A7 (S100A7) Defensin-b 4 (DEFB4; hBD-2)
NM_002963 NM_004942
1.56 1.50
Cystatin C (CST3) Adrenomedullin (ADM)
NM_000099 NM_001124
1.07 1.06
RNase 7 (RNASE7) Mucin 5 (MUC5B)
NM_032572 S80993
1.02 0.97
Defensin-b 103A (DEFB103A; hBD-3) Hepcidin antimicrobial peptide (HAMP)
NM_018661 NM_021175
0.92 0.90
S100A8
Defensin-b 1 (DEFB1; hBD-1) Azurocidin 1 (AZU1)
NM_005218 NM_001700
0.89 0.82
S100A9
Lysozyme 2 (LYZL2) Defensin-b 123 (DEFB123)
ENST00000280633 NM_153324
0.82 0.82
Seminal plasmin (PYY2) Dermcidin (DCD)
NM_021093 NM_053283
0.87 0.81
DEFB103A
GAPDH
Treatment time (h)
b 0
6
C C I
12
24
48
72
C I
C I
C I
C
I
GAPDH
Treatment time (h) 0
6
12
24
48
72
C C I C I C I C I C I
The fold difference in AMP mRNA expression in IL-1a-stimulated HaCaT cells normalized to nonstimulated cells is shown.
LCN2 SLPI S100A7
IL-1a (Figure 1c). Expression of AMPs was generally maximal in response to 10 mg/ml IL-1a.
GAPDH
c AMP proteins upregulated by IL-1a The effect of IL-1a on AMP expression in HaCaT cells was investigated at the protein level by western blotting (Figure 2). IL-1a increased the protein expression of lipocalin 2, S100A8 and S100A9, SLPI and S100A7, whereas hBD-2 protein was unaffected (data not shown).
IL-1α (ng /ml) 0
S100A8 S100A9
0.1
1
10
IL-1α (ng /ml)
100
0
0.1
1
10
100
LCN2 SLPI S100A7
DISCUSSION Many AMPs are generally expressed in mammalian squamous mucosal epithelium and epidermis during apparent health, and serve as the biological barrier against infecting bacteria, fungi and virus, and contribute to innate immunity.7 Some peptides are constitutively expressed and other peptides are regulated by microbes and proinflammatory cytokines including IL-1b, TNF-a and interferon-g (IFN-g). In the present study, mRNAs for 18 AMP genes appeared to be expressed by human skin-derived keratinocytes (HaCaT cells). Of these, six AMP genes (LCN2, S100A8, S100A9, SLPI, S100A7, DEFB4) and five AMP proteins (LCN2, S100A8, S100A9, SLPI and S100A7) were upregulated within 72 h by IL-1a in a dose- and time-dependent manner. In response to IL-1a, the keratinocyte appears to launch a robust response of AMPs. The AMP gene most upregulated by IL-1a is LCN2. Secreted from lacrimal gland and a major component in tears, LCN inhibits antimicrobial growth through binding to iron siderophores.21 In human keratinocytes, LCN2 expression is also upregulated by IL-1b, TGF-a and insulin-like growth factor I.22 In the present study, IL-1a increased the expression of S100A8- and S100A9-specific mRNAs and proteins, which complex spontaneously to form calprotectin. Calprotectin is upregulated in infected and inflamed keratinocytes,10,12,23,24 and appears to protect the cytoplasm of keratinocytes against invading bacteria.10 Calprotectin complex may also function in the regulation of cell cycle (Wang et al, in preparation, 2007).25–28 We found that inducers of keratinocyte differentiation, such as phorbol myristate acetate,13 IL-1a and Immunology and Cell Biology
GAPDH
GAPDH
Figure 1 Verification of AMP mRNA expression in IL-1a-stimulated HaCaT cells by RT-PCR and northern blotting. (a) Subconfluent HaCaT cells were incubated with IL-1a (10 ng/ml) for 24 h. After RNA isolation, the expression of LCN2, S100A9, S100A8, SLPI, S100A7 and GAPDH mRNAs were analyzed by RT-PCR and northern blotting. The expression of DEFB4, CST3, ADM, RNASE7, MUC5B, DEFB103A, HAMP, DEFB1 and AZU1 mRNAs were analyzed by RT-PCR. The expression of S100A8 and S100A9 mRNAs were confirmed by 10 individual experiments, LCN2 and SLPI by four experiments, and other AMP expression was verified by three experiments. (b) Time course of AMP mRNA expression in HaCaT cells in response to IL-1a. Cells were harvested at the times indicated up to 72 h in the presence (I, IL-1a) or absence (C, control) of 10 ng/ml IL-1a, RNA was harvested and expression of AMPs-specific mRNAs was estimated by northern blotting. (c) Cells were incubated for 24 h in the presence of concentrations of IL-1a up to 100 ng/ml, RNA was harvested, and AMP mRNA levels were estimated by northern blotting.
calcium,3 and proinflammatory cytokines (IL-1b, TNF-a, IL-6; unpublished data) also upregulate S100A8/S100A9 expression in human gingival keratinocytes. In human skin keratinocytes, which do not express calprotectin in the absence of inflammation or wound healing, calprotectin expression is upregulated by IL-1b, TNF-a, IFN-g and IL-1a (this study).14 S100A7 (psoriasin) is also a calcium-binding protein that appears to show zinc-dependent antimicrobial activity, and is expressed in the suprabasal layers of healthy skin.29 The expression of S100A7 in human keratinocytes is upregulated by
IL-1a regulates AMP expression M Bando et al 535
Cont.
Table 3 RT–PCR primers and conditions
IL-1α 25 kD
Lipocalin 2
S100A8
14 kD
S100A9
8 kD
Gene
S100A7
11.4 kD
Figure 2 Effect of IL-1a on the expression of lipocalin 2, S100A7, S100A8 and S100A9 proteins in HaCaT cells. Sub-confluent cells were incubated with IL-1a (10 ng/ml) for 48 h. Cells were sonicated to disrupt, and 15 mg (or 50 mg for SLPI) of HaCaT cell protein in each sonicate fraction was analyzed for lipocalin 2, S100A7, S100A8, S100A9 and SLPI by western blotting. The expression of AMP proteins was confirmed by at least two replicate experiments.
TNF-a,29
Escherichia coli, IL-1b or and is also increased in welldifferentiated keratinocytes from patients with psoriasis.30 The present study shows that S100A7 is slightly upregulated by IL-1a, suggesting that, like calprotectin, expression in keratinocytes is also regulated by modulators of keratinocyte differentiation and proinflammatory cytokines, and perhaps microbes. SLPI is produced by epithelial cells, neutrophils and macrophages, and detected in saliva, tears and breast milk.15 SLPI has broadspectrum antimicrobial activity and has been implicated as a major anti-HIV mechanism in saliva.31–34 SLPI is constitutively expressed in human keratinocytes and upregulated by IL-1b, TNF-a, epidermal growth factor and IL-1a (this study),22,35 and is also increased by exposure of gingival keratinocytes to HIV.32 Hence, SLPI expression appears to be modulated similarly. The human b-defensins are major cationic AMPs expressed in epidermal, mucosal and gingival keratinocytes with antimicrobial activity against Gram-positive and -negative bacteria, and Candida.1,2,36,37 In the present microarray analysis, HaCaT cells expressed DEFB4 (hBD-2), DEFB103A (hBD-3), DEFB1 (hBD-1) and DEFB123. Of these defensin genes, only hBD-2 appeared to be differentially regulated in response to IL-1a at the mRNA level, but differences in immunoreactive peptide levels were not apparent (data not shown). hBD-2 expression is increased by IL-1a, IL-1b, TNF-a and bacteria in human epidermal and epithelial cells.5,6,17,18,38 hBD-3 is upregulated by TNF-a, IFN-g, but not by IL-1b.6,37,38 hBD-1 is constitutively expressed in keratinocytes and not markedly regulated by proinflammatory cytokines (IL-1b and TNF-a) or bacteria.4,6 Unlike other MAPs, hBD-1 and -3 expression in HaCaT cells was unaffected by IL-1a. Like hBD-1, the AMPs, ADM and RNase 7 are expressed by HaCaT cells, but are not apparently regulated by IL-1a. In gut epithelial cells, however, ADM is upregulated by IL-1a,39 while ADM expression is increased by TNF-a and LPS in oral, but not in skin keratinocytes.40 RNase 7 is expressed in keratinocytes of healthy skin,7,41 but is upregulated by IL-1b, IFN-g and bacteria.41 Also expressed in HaCaT cells and unaffected by IL-1a are CST3, HAMP, AZU1 and DCD. HAMP,
PCR
PCR product
cycle
size (bp)
ADM
For: 5¢-ATGAAGCTGGTTTCCGTC-3¢
33
506
AZU1
Rev: 5¢-TGTGGCTTAGAAGACACC-3¢ For: 5¢-GACTGGATCGATGGTGTTCTC-3¢
40
235
CST3
Rev: 5¢-CAGAGGAGAGATCGGCTTCTT-3¢ For: 5¢-AGATCGTAGCTGGGGTGAACT-3¢
33
150
DEFB1
Rev: 5¢-GCACAGCGTAGATCTGGAAAG-3¢ For: 5¢-TGAGTGTTGCCTGCCAGTCGC-3¢
38
191
DEFB4
Rev: 5¢-CTTGAATTTTGGTAAAGATCG-3¢ For: 5¢-CCAGCCATCAGCCATGAGGGT-3¢
38
255
Rev: 5¢-GGAGCCCTTTCTGAATCCGCA-3¢ DEFB103A For: 5¢-CCTTTTCATCCAGTCTCAGCG-3¢
40
213
GAPDH
Rev: 5¢-GCGTCGAGCACTTGCCGATCT-3¢ For: 5¢-TCCACCACCCTGTTGCTGTA-3¢
30
451
HAMP
Rev: 5¢-ACCACAGTCCATGCCATCAC-3¢ For: 5¢-CTGACCAGTGGCTCTGTTTTC-3¢
40
192
LCN2
Rev: 5¢-GGGCAGGTAGGTTCTACGTCT-3¢ For: 5¢-TGTCACCTCCGTCCTGTTTAG-3¢
30
226
11.7 kD
SLPI
Primer
Rev: 5¢-TCTCCCGTAGAGGGTGATCTT-3¢ MUC5B2
For: 5¢-TGCAATCAGCACTGTGACATTGAC-3¢ Rev: 5¢-TTCTCCAGGGTCCAGGTCTCATTC-3¢
40
243
RNASE7
For: 5¢-TTTGGCTGACCTTCAATTCC-3¢ Rev: 5¢-TCTTGGGGATAAGCATCTGG-3¢
40
199
S100A7
For: 5¢-TGCTGACGATGATGAAGGAG-3¢ Rev: 5¢-ATGTCTCCCAGCAAGGACAG-3¢
30
151
S100A8
For: 5¢-GCTGGAGAAAGCCTTGAACTC-3¢ Rev: 5¢-CCACGCCCATCTTTATCACCA-3¢
30
232
S100A9
For: 5¢-TCGCAGCTGGAACGCAACATA-3¢ Rev: 5¢-AGCTCAGCTGCTTGTCTGCAT-3¢
30
213
SLPI
For: 5¢-CAGAGTCACTCCTGCCTTCAC-3¢ Rev: 5¢-CTCTGGCACTCAGGTTTCTTG-3¢
35
180
normally expressed by cells in the liver, is upregulated by IL-6 in hepatocytes,42 but expression and regulation in epithelia are not known. DCD is constitutively expressed by eccrine glands and the product is a principal AMP in sweat.7 AZU1, an cationic antimicrobial protein, is expressed in inflammation and induced by IL-1b and TNF-a in corneal epithelial cells,43 but AZU1 expression was unaffected by IL-1a in HaCaT cells. These results show that some AMPs including ADM, RNase 7, HAMP, DCD and AZU1 and so on appear to be differentially expressed in a tissue-, proinflammatory mediatorand keratinocyte differentiation regulator-specific manner. While proinflammatory cytokines and microbes generally regulate AMP expression, there is little information about the biology of IL-1a in keratinocytes. We found that nonstimulated HaCaT cells constitutively express IL-1a mRNA (data not shown) and show low levels of expression of some AMPs. IL-1a is also constitutively expressed by oropharyngeal keratinocytes and upregulated in oral mucosal inflammation and lichen planus.44,45 We now show that IL-1a modulates keratinocyte immune molecules including AMPs, in addition to cytokines and chemokines. The data therefore suggest that IL-1a regulates cell autonomous immunity in keratinocytes. METHODS Cell culture An immortalized epidermal human keratinocyte cell line, HaCaT46 was supplied by Dr N Fusenig (German Cancer Research Center), inoculated at Immunology and Cell Biology
IL-1a regulates AMP expression M Bando et al 536 5200 cells/cm2 and cultured in Dulbecco’s minimal essential medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin for 5 days. At 90% confluence, HaCaT cells were further cultured for 24 h (microarray, northern blot and RT-PCR analyses) or 48 h (western blot analysis) with or without 10 ng/ml of IL-1a (Wako, Osaka, Japan), and used to examine AMP gene and protein expressions.
RNA isolation and microarray analysis Total RNA was isolated from IL-1a-stimulated and nonstimulated HaCaT cells using an RNeasy kit (Qiagen, Valencia, CA, USA). Relative purity was examined using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). For each condition, RNA expression analysis was performed using the Whole Human Genome Oligo Microarray (Agilent Technologies). This microarray chip contains 41 059 oligonucleotide probes for known and unknown genes. First strand cDNA was synthesized from 10 mg total HaCaT RNA using the SuperScript Indirect cDNA Labeling System (Invitrogen, San Diego, CA, USA) and labeled for 16 h in the dark with cyanine-3 or cyanine-5 (Cy3, Cy5; MonoReactive Dye Pack; GE Healthcare Life Sciences, Piscataway, NJ, USA). The labeled cDNA was purified using a MiniElute Reaction Cleanup Kit (Qiagen) and denatured at 981C for 3 min. The denatured labeled cDNA was mixed with deposition control targets for chip normalization in hybridization buffer, applied to two microarray slides in a hybridization chamber, and hybridized at 601C for 17 h according to standard protocols from Agilent Technologies. On one chip, Cy3-labeled cDNA derived from nonstimulated cells (control) and Cy5-labeled cDNA from IL-1a-stimulated cells were hybridized. On a second chip, dye-swapped samples were hybridized; the Cy5-labeled control cDNA and Cy3-labeled IL-1a-stimulated cDNA were hybridized. After washing in SSC-Triton-X washing solution, the microarray slide was scanned in a microarray slide scanner to determine the fluorescence intensities of hybridized Cy3- and Cy5-labeled cDNAs. Intensity data for the two chips per comparison experiment were normalized by dye swap and flag treatments and analyzed with GeneSpring 7.0 (Silicon Genetics, Redwood City, CA, USA). Only gene expression showing fluorescence intensity greater than 100 was analyzed (Tables 1 and 2). Genes expressed more than 2-fold and less than 0.5-fold of control by IL-1a treatment were listed in Table 1 as up- or downregulated. AMP genes expressed in HaCaT cells are shown in Table 2.
RT-PCR and northern blotting To confirm expression of select AMP genes, HaCaT cell RNA samples were analyzed by microarray and RT-PCR. Briefly, cDNA was synthesized from 1 mg of the RNA sample using cDNA Synthesis Kit 1st Strand (Life Sciences Inc., St Petersburg, FL, USA). The cDNA was added to the PCR mixture containing primers of AMPs and then amplified 30–40 cycles using the following conditions: denature at 941C for 1 min, anneal at 55–641C for 1 min, followed by extension at 721C for 1 min. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing 0.1 mg/ml ethidium bromide. The primers and PCR cycle numbers used in this study are shown on Table 3. For genes (LCN2, S100A8, S100A9 and SLPI) of interest that increased more than twofold in microarray analysis and for the S100A7 gene, expression was further confirmed by northern blotting. Briefly, 15 mg of total RNA were electrophoretically separated on a 6% formaldehyde and 1% agarose gel, and transferred to a Hybond N+ membrane (GE Healthcare Bio-Science Co., Piscataway, NJ, USA). The probes were prepared by PCR and labeled with [a-32P]dCTP using the BcaBest Labeling Kit (TaKaRa Bio Inc., Otsu, Shiga, Japan). Prehybridization and hybridization were performed at 421C for 2 and 12 h, respectively. The autoradiography bands were analyzed using the BAS 2000 Bio-Imaging Analyzer (Fuji Photofilm Co., Tokyo, Japan).
Western blotting The cultured HaCaT cells were suspended in 10 mM Tris-HCl buffer (pH 7.4) with a protease inhibitor cocktail and disrupted by sonication. The expression of AMP proteins in the sonicate were analyzed by western blotting using each AMP antibody according to a modification of a method described previously.47 Briefly, 15 or 50 mg of sonicate protein was electrophoretically separated on 12.5% polyacrylamide gels and electrically transferred to PVDF membranes (Immobilon-P, Millipore, Bedford, USA). After blocking in 5% skim milk, Immunology and Cell Biology
membranes were reacted with rabbit anti-human AMP antibodies against lipocalin 2, S100A7, S100A8, S100A8, SLPI and hBD-2 for 3–4 h. Membranes were then reacted with horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako Cytomation Japan, Kyoto, Japan) for 1.5 h at room temperature, developed with the ECL Western Blocking Detection System (GC Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and exposed to Hyperfilm-ECL (GC Healthcare Bio-Sciences Corp.). The antibodies against lipocalin 2 and SLPI were purchased from R&D Systems Inc. (Minneapolis, MN, USA), S100A7 antibody from Acris Antibodies GmbH (Hiddenhausen, Germany), and hBD-2 antibody from Alpha Diagnostic International (San Antonio, TX, USA). The S100A8/S100A9 antibody was kindly supplied by Dr MK Fagerhol (Ullevaal University Hospital, Oslo, Norway).47
ACKNOWLEDGEMENTS This study was supported in part by Grants-in-Aid (#15592190) for Scientific Research from the Japan Society for the Promotion of Science and by NIH Grants R01DE11831 and R01DE15503 to MCH.
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Immunology and Cell Biology
